Abstract

Background

Essential hypertension is a prevalent complex polygenic disease and a major risk factor for cardiovascular disease, the leading cause of death in developed countries. Because of its complex and multifactorial nature, its genetic determinants still remain largely unknown. The Dahl saltsensitive hypertensive rat model exhibits impaired sodium handling, which is hypothesized to play a key role in the pathophysiology of polygenic hypertension. Thus, genes associated with renal regulation of salt and water balance are a priori likely candidates for a causative role in hypertension pathogenesis. The functional properties and renal-specific expression of the recently characterized AngII/AVP receptor suggest a putative modulator role in tubular sodium and fluid reabsorption. Based on these observations, we investigated the potential involvement of the AngII/AVP receptor in salt-sensitive hypertension.

Materials and Methods

We performed cosegregation analysis of the AngII/AVP receptor locus with salt-sensitive hypertension in an F2 (Dahl S × Dahl salt-resistant [R]) hybrid male cohort characterized for blood pressure by radiotelemetry after 8 weeks of high salt challenge. Further molecular analysis was done to identify putative AngII/AVP receptor molecular variants that could account for the AngII/AVP receptor involvement in salt-sensitive hypertension pathogenesis.

Results

The AngII/AVP receptor was mapped to rat chromosome 1, 1.7 cM centromeric to the D1Rat188 marker by radiation hybrid mapping analysis. Quantitative trait locus (QTL) analysis detected a highly significant linkage of the AngII/AVP receptor locus with high blood pressure (LRS = 13.8, p = 0.0002). Molecular characterization of the Dahl S and Dahl R AngII/AVP receptor cDNAs revealed two amino acid substitutions in the Dahl S AngII/AVP receptor (N119S, C163R) when compared to the Dahl R AngII/AVP receptor. These mutations are associated with an increased receptor affinity for both ligands (AVP and AngII) and an enhanced Gs-coupling by the receptor resulting in increased activation of adenylate cyclase with concomitant increase in cAMP production.

Conclusions

The observed molecular dysfunction in the Dahl S AngII/AVP receptor is consistent with increased tubular sodium and fluid reabsorption observed in Dahl S rats. Interestingly, the AngII/AVPr locus is within the narrowed chromosome 1 QTL region for blood pressure detected in different rat intercross linkage analyses. Altogether, the data strongly suggest that the AngII/AVP receptor is a hypertension susceptibility gene in the Dahl S rat model, as well as raises the hypothesis that it too underlies the chromosome 1 blood pressure QTL identified in other hypertension rat models.

Introduction

The kidney plays a primary role in hypertension pathogenesis in the Dahl salt-sensitive hypertensive rat model (1–3). Transplant studies have shown that the donor kidney determines the blood pressure phenotype in the bilaterally nephrectomized recipient (3). A similar situation has also been described in human renal transplant patients (3). These facts validate the analysis of genes expressed preferentially within the kidney as logical candidates for hypertension-susceptibility genes. Because the AngII/AVP receptor is prominently expressed in renal epithelial cells (4,5), and both AngII and AVP hormones are both known to modulate tubular sodium and fluid reabsorption (6–14), we investigated whether this receptor could have a potential role in hypertension pathogenesis. The AngII/AVP receptor is coupled to adenylate cyclase and responds with equal sensitivity to AngII and AVP (4). Pharmacologic characterization defines this dual receptor as a novel AT1/V2 type of receptor (5). These receptor properties, in conjunction with its renal immunocytochemical distribution to the outer medullary thick ascending limb tubules and inner medullary collecting ducts (5), suggest that the AngII/AVP receptor could play a prominent role in renal tubular sodium and fluid reabsorption. Thus, it is intriguing to hypothesize that this dual receptor could mediate the coordination of two distinct blood pressure regulatory systems.

To assess the putative role of the AngII/AVP receptor in hypertension pathogenesis, we performed genetic and molecular analyses in the Dahl S/Dahl R rat genetic model of salt-sensitive hypertension. The results reported here strongly suggest that the AngII/AVP receptor is a hypertension-susceptibility gene in the Dahl S rat model, thus providing impetus for the future investigation of its potential role in human essential hypertension.

Materials and Methods

Chromosomal Localization of the AngII/AVP Receptor Gene

To localize the AngII/AVP receptor gene within the rat genome, we conducted a radiation hybrid panel screening on a rat panel (Rat/Hamster RH panel RH07) obtained from Research Genetics (Huntsville, AL, USA). A 479-bp polymerase chain reaction (PCR) fragment (upstream primer: 5′-gtt-aca-gag-ctg-agg-gcc-tg-3′; downstream primer: 5′-cct-gga-aac-cac-act-cac-ct-3′) from the 5′ regulatory region of the rat AngII/AVP receptor gene was utilized as indicator product. The data were then tested against the existing framework maps. The results assign the AngII/AVP receptor gene to chromosome 1, 14.6 cR from D1Rat111 with a LOD = 24.673. This places the AngII/AVP receptor gene approximately 1.7 cM centromeric to D1Rat188 (Table 1).

Functional Analysis

The Dahl S and Dahl R AngII/AVP receptor cDNAs were subcloned directionally (5′ to 3′) into the NheI site of the pMAMNeo expression vector (Clontech, Palo Alto, CA, USA). Stable Cos1 neomycin-resistant transfectants were developed as described (4). 125I-AngII and 3H-AVP binding experiments were preformed essentially as described (4) on intact Cos1 pMAM-Dahl S AngII/AVPr and Cos1 pMAM-Dahl R AngII/AVPr cell transfectants. Specific binding was determined as the difference between the total radioactivity bound to cells and the radioactivity bound to blanks containing 1 µM of AngII or 10 µM of AVP. AngII- and AVP-dependent stimulation of cAMP accumulation was assayed on C127 cell membranes after transient transfection of C127 cells (mouse mammary tumor cell line, ATCC) with the pMAM-Dahl S AngII/AVPr and the pMAM-Dahl R AngII/AVPr expression vectors. For this purpose, the pMAM-Dahl S AngII/AVP receptor and the pMAM-Dahl R AngII/AVP receptor expression vectors (60 µg each) were introduced into C127 cells (107 cells) via lipofectinmediated transfection. After 5 days in culture, cells were harvested and a crude membrane preparation was obtained as described (19). 10 µg of Dahl S and Dahl R AngII/AVP receptor expressing membranes were exposed to 0.1 µM of AVP and 0.1 µM of AngII at 25°C for 20 min with 0, 50, and 150 mM of NaCl, in comparison to basal control (no hormone added) in an incubation buffer containing 40 mM of Tris-HCl (pH 7.6), 5 mM of MgCl2, 1 mM of EDTA, 2 mM of isobutylmethylxanthine, 3 mM of ATP, 20 mM of phosphocreatine, 5 U/ml of creatinephosphokinase, 0.1 µM of GTP, and 0.1% BSA. The level of cAMP was determined by radioimmunoassay according to manufacturer’s specifications (Amersham, Piscataway, NJ, USA).

Results

Intercross Linkage Analysis

To assess the potential genetic contribution of the AngII/AVP receptor gene to salt-sensitive hypertension susceptibility, we performed an intercross linkage analysis on 105 F2 (Dahl S ♂ × Dahl R ♀) hybrid male rats phenotyped for BP by radiotelemetry after 8 weeks of high salt (8% NaCl) challenge (15). We localized the AngII/AVP receptor gene to rat chromosome 1, 1.7 cM centromeric to the D1Rat188 marker (see Table 1) by radiation hybrid mapping analysis. Thus, 105 F2 male hybrids were genotyped at nine informative markers that spanned the AngII/AVP receptor locus on chromosome 1. As seen in Table 1, LRS and p values peaked at D1Rat188 (the closest marker, 1.7 cM to the AngII/AVP receptor locus) for SBP (LRS = 13.8, p = 0.00020), DBP (LRS = 8.6, p = 0.00336), and MAP (LRS = 12.4, p = 0.00043). These results fulfill the first of four criteria needed to identify a hypertension susceptibility gene (15,20): 1) association of the putative hypertension susceptibility gene with hypertension in validated genetic animal models or human hypertensive patients, 2) identification of a functionally significant structural mutation in the relevant gene, 3) concordance of the observed molecular dysfunction with a pathophysiologic mechanism logical to the hypertension pathogenesis, and 4) delineation of the mechanistic role in an in vivo model. Based on a permutation test performed on the 105 informative progeny that established the minimum LRS value for highly significant as equal to 12.2, the AngII/AVPr locus exhibits a highly significant linkage with SBP (LRS = 13.8). Analysis for interaction with α1 Na,K-ATPase, bumetanide-sensitive Na,K,2Cl-cotransporter and thiazide-sensitive Na, Cl-cotransporter loci were negative. As shown in Table 1, the AngII/AVP receptor locus accounts for 12% of the SBP variance.

Hormone-dependent stimulation of cAMP accumulation in Dahl S and Dahl R AngII/AVP receptors expressed in C127 cells. Ten micrograms of Dahl S and Dahl R AngII/AVP receptor expressing membranes were exposed to 0.1 µM of AVP (a) or 0.1 µM of AngII (b) at 25°C for 20 min in the presence of 0, 50, or 150 mM of NaCl, in comparison to basal control (no hormone added). Each experiment was performed in triplicate. Values are presented as mean ± standard deviation.

Discussion

Our studies have detected a modulator effect of sodium on G-protein coupling, a unique feature of the AngII/AVP receptor function that could have a significant impact on modulation of receptor function under physiologic conditions. Whereas AVP-dependent activation of cAMP accumulation (presumably via Gs-receptor coupling) is minimally affected by sodium in the wild-type (Dahl R) receptor, the AngII-dependent activation of cAMP accumulation is dramatically influenced by sodium in both Dahl S and Dahl R receptors resulting in a dichotomous sodium effect. Inhibition of AngII-induced cAMP accumulation (presumably via Gi-receptor coupling) is observed in the absence of sodium, whereas stimulation of AngII-induced cAMP accumulation (presumably via Gs-receptor coupling) is observed in the presence of sodium (see Fig. 3).

Sodium modulation of receptor function is not without precedent. Allosteric regulation of α2-adrenergic receptor ligand binding by sodium has been reported (21) in which the allosteric effects of sodium have been found to be maximal at 40 mM (21). This is similar to the maximal effect of sodium on AngII/AVP receptor function detected at 50 mM (see Fig. 3). Altogether, the data suggest that the sodium modulation of AngII/AVP receptor function is a biologically significant phenomenon that contributes to susceptibility to salt-sensitive hypertension in the Dahl S rat model. Furthermore, we hypothesize that this receptor could be a key component of a sodium-sensing gene network underlying physiologic regulation of sodium homeostasis. It is important to note that the AngII/AVP receptor is broadly expressed in neurons of the central nervous system, including those regions thought to be involved in central control of blood pressure and salt appetite (22,23). However, additional molecular, cellular, and physiologic studies are necessary to test this hypothesis.

The differential effects of sodium on AngII- and AVP-induced cAMP accumulation indicate that the AngII/AVP receptor mediates AngII and AVP stimulation via two distinct effector pathways. Moreover, it also documents alternative use of guanine nucleotidebinding regulatory proteins by a single receptor as a mechanism for diversifying receptor function. The sodium-dependent “switching” of the AngII/AVP receptor from Gi to Gs coupling observed in our studies, resembles the PKA-dependent “switching” from Gs to Gi coupling recently reported in the α2-adrenergic receptor (24), thus corroborating the existence of multiple G-protein coupling “switching” mechanisms.

The localization of the amino acids substitutions within the Dahl S receptor suggests that they may differentially influence receptor function. The N119S substitution in the H2–H3 intracellular loop (see Fig. 1b) could affect GS coupling because of its proximity to the putative GS activation domain (H4–H5 intracellular loop, see Fig. 1b). In contrast, the C163R substitution in the H3–H4 extracellular loop (see Fig. 1b) might influence AVP and AngII binding due to its putative effects on the secondary structure of the extracellular receptor domains.

Our results demonstrate the existence of a “hyperfunctioning” AngII/AVP receptor present in Dahl salt-sensitive rats exhibiting increased affinity for both ligands (AVP and AngII) and enhanced GS coupling resulting in increased activation of adenylate cyclase. Analogous dysfunctional behavior has been reported for the constitutively activated mutation of the luteinizing hormone receptor in familial male precocious puberty (25) and in thyrotropin receptor involved in thyroid adenomas (26). Thus, for any given level of ligand (AVP or AngII) concentrations, the “hyperfunctional” Dahl S AngII/AVP receptor will effectively produce a higher level of cAMP accumulation compared with the Dahl R receptor. Intuitively, we hypothesize that this AngII-AVP “hyperresponse” could contribute to increased tubular sodium and fluid reabsorption in Dahl S rats, a characteristic pathophysiology observed in this genetic hypertensive rat model (3). Our hypothesis is substantiated by the prominent expression of the AngII/AVP receptor in the thick ascending limb of the loop of Henle and collecting ducts (5).

Most importantly, the selective AngII-dependent sodium hyperresponsiveness of the dual AngII/AVP receptor is concordant with studies in humans demonstrating the central role of the renin-angiotensin system (RAS) in salt-sensitive blood pressure response (38). Altogether the data provide a compelling basis and strategic focus for investigating the role of the AngII/AVP receptor in human hypertension.